Analysis method using reporter (label) intermolecular interaction

ABSTRACT

The present invention provides, in the use of the energy transfer phenomenon in a detection system for the measurement of the concentration of a material, a method for controlling the spatial arrangement of an energy donor and an energy acceptor in a reaction complex in order to carry out the measurement with good precision and high sensitivity, and a measurement system using the method.  
     Two types of material having affinity for the subject material to be measured are respectively labeled with a combination of reporters that give rise to energy transfer; those that have been thus obtained by labeling these materials are each further labeled with materials that have weak affinity for each other to give reagents, which are then mixed with a sample to give the reaction complex. Each of the materials is brought into spatial proximity by binding based on the affinity among the materials having weak affinity for each other in the reaction complex, and since that condition is stably maintained, a more efficient energy transfer occurs.

TECHNICAL FIELD

[0001] The present invention relates to a method of analyzing materialsby means of interaction between reporters. In particular, it relates toa high sensitivity analytical method in which the interaction betweenreporters increases as the reporters are brought in proximity to eachother.

BACKGROUND ART

[0002] Intensive research has been carried out into, for example,increasing the sensitivity and simplifying the measurement systems forthe analysis and measurement of specific materials in various fieldssuch as clinical diagnosis, food hygiene, and environmental hygiene. Inparticular, immunoassays employing an antigen-antibody reaction are morewidely employed thereamong, for reasons of sensitivity and specificity.The sandwich enzyme immunoassay (EIA) method can be cited as arepresentative immunoassay method.

[0003] In sandwich EIA, of two types of antibody for a subject materialto be measured, one is immobilized on a solid phase; this solid phase,the other antibody which is labeled with an enzyme, and a test sampleare mixed together and reacted; after removing unreacted enzyme-labeledantibody by washing, an enzyme substrate solution is added and theamount of immunocomplex generated is measured by the enzyme activity.Since this method has good reproducibility and, furthermore, the antigenconcentration can be measured with high sensitivity, it is widelyemployed. However, the sandwich method has the problem that acomplicated and time-consuming washing operation for removing unreactedlabeled antibody is necessary, etc.

[0004] In order to solve this problem, in recent years immunoassaymethods employing energy transfer phenomena such as fluorescence energytransfer (FRET), bioluminescence energy transfer (BRET), andchemiluminescence energy transfer (CRET), or intermolecular interactionssuch as the complementation of enzyme activity of enzyme molecules ofwhich a part has been deleted or altered have been noteworthy.Fluorescence energy transfer is a phenomenon observed between certainspecific fluorescent materials; in a case where the fluorescencespectrum of a fluorescence energy donor and the excitation lightspectrum of a fluorescence energy acceptor overlap and, moreover, wherethe distance between these two molecules approaches 10 nm or less, thefluorescence energy of the fluorescence energy donor transfers to theacceptor, and luminescence of the fluorescence energy acceptor isobserved.

[0005] Bioluminescence energy transfer is a phenomenon observed betweena fluorescent material and an enzyme, such as firefly luciferase, thatcatalyses bioluminescence; in a case where the luminescence spectrum ofthe bioluminescence enzyme and the excitation light spectrum of aluminescence energy acceptor overlap and, moreover, where the distancebetween these two molecules approaches 10 nm or less, the luminescenceenergy of the bioluminescence energy donor transfers to the acceptor,and fluorescence of the luminescence energy acceptor is observed.

[0006] Chemiluminescence energy transfer is a phenomenon observedbetween a fluorescent material and an enzyme, such as a peroxidase, thatcatalyses chemiluminescence; in a case where the luminescence spectrumof the chemiluminescence enzyme and the excitation light spectrum of aluminescence energy acceptor overlap and, moreover, where the distancebetween these two molecules approaches 10 nm or less, the luminescenceenergy of the chemiluminescence energy donor transfers to the acceptor,and fluorescence of the luminescence energy acceptor is observed.

[0007] The complementation of enzyme activity of enzyme molecules ofwhich a part has been deleted or altered is a phenomenon typified by theβ-galactosidase Δα mutant in which the α site has been deleted, and theΔω mutant in which the ω site has been deleted; by themselves Δα and Δωhave reduced β-galactosidase enzyme activity, but when the two approacheach other the β-galactosidase enzyme activity increases as a result ofassociation. Several examples have been reported so far in which suchenergy transfer phenomena and such complementation of enzyme activity ofenzyme molecules of which a part has been deleted or altered are appliedto immunoassay and the detection of protein-protein interaction.

[0008] Among these, as an example of the application of the energytransfer phenomena to an immunoassay there can be cited a methodreported by Toyobo Co., Ltd. (JP, A, 10-319017). In this method,materials A and B that have the capacity to bind to a subject material(X) to be measured are labeled with materials F1 and F2 respectively,which have a fluorescence energy donor-acceptor relationship; A-F1, X,and B-F2 are mixed and reacted for a fixed time, and after forming acomplex F1-A:X:B-F2, without carrying out a washing operation, theamount of F1-A:X:B-F2 is measured as the efficiency of fluorescenceenergy transfer from F1 to F2.

[0009] However, the actual subject material to be immunoassayed(antigen) is often a high molecular weight protein and, furthermore,when two types of monoclonal antibody are used, etc., since the twoepitopes are spatially separated, even if the reaction complexF1-A:X:B-F2 is formed, F1 and F2 in the F1-A:X:B-F2 complex are not inspatial proximity, and efficient energy transfer cannot take place. As aresult, there is the problem that the sensitivity of the measurement islow.

[0010] According to Förster's theory of fluorescence energy transfer,the efficiency of fluorescence energy transfer is inversely proportionalto the sixth power of the distance between the donor and acceptor (Anal.Biochem, 218, 1-13, 1994). Thus, when applying the FRET, BRET, CRET,etc. energy transfer phenomena to an immunoassay, it is necessary toestablish a technique that always arranges the luminescence energy donorand acceptor in fixed positions within this type of immunocomplex.

[0011] In the application of FRET to immunoassay, the method of Ueda etal. (JP, A, 10-78436) can be cited as an example in which the spatialarrangement of the energy donor and acceptor is carried out well. Inthis method, antibody VL and VH fragments are labeled with fluorescenceenergy donor and acceptor and, in the presence of an antigen,fluorescence energy transfer takes place only when a stable three-wayassociation of antigen, VL, and VH is formed, and by measuring thefluorescence intensity ratio of the fluorescence energy donor andacceptor, the antigen concentration is measured. It has beendemonstrated (Anal. Biochem, 289, 77-81, 2001) that this measurementmethod employing VL and VH can measure the antigen concentration in BRETalso.

[0012] This method, which combines, with fluorescence energy transfer,the property of VL and VH antibody fragments always being within 5 nmwhen an antigen is present, is an exceptionally unique immunoassaymethod, but since for many of the antibodies present in nature VL and VHhave the property of associating even in the absence of an antigen, thisprinciple cannot be applied to all antibodies. As a result, it cannot beput to practical use.

[0013] Furthermore, a method in which the complementation of enzymeactivity of enzyme molecules of which a part has been deleted or alteredwas applied to monitoring protein-protein interaction has been reportedby Rossi et al. (Proc. Natl. Acad. Sci. USA, 94, 8405-8410, 1997). Inthe method of Rossi et al., expression vectors of the proteins FRAP andFKBP12, for which dimerization is induced by rapamycin, fused with Δαand Δω β-galactosidases respectively were introduced into mammaliancells; when rapamycin was added to these chimeric protein-expressingcells, dimerization of the FRAP and FKBP12 proteins took place due tothe rapamycin, and as a result rapamycin concentration-dependentβ-galactosidase activity could be measured.

[0014] This method is an extremely good method for carrying outmeasurement of a material in viable cells, but it is an extremelyspecial example in which the low molecular weight rapamycin-induceddimerization of FRAP and FKBP is utilized. When using this kind ofenzyme complementation in the detection of an immunocomplex inimmunoassay, in the same way as immunoassay methods employing energytransfer such as FRET, a technique that always arranges reporterproteins in fixed positions is also necessary.

[0015] Therefore, when intermolecular interactions such as an energytransfer phenomenon like fluorescence energy transfer (FRET) orluminescence-fluorescence energy transfer (BRET) and the complementationof enzyme activity of enzyme molecules of which a part has been deletedor altered are used in material concentration measurement detectionsystems, in order to carry out measurement with good precision and highsensitivity, the present invention provides a method for controlling thespatial arrangement of the energy donor and energy acceptor in thereaction complex, or the spatial arrangement of the complementationreaction donor and the complementation reaction acceptor, and ameasurement system to which the method is applied.

DISCLOSURE OF INVENTION

[0016] We first labeled each of two types of materials having affinityfor a subject material to be measured (materials that directly affinitybind to the subject material to be measured) with a fluorescence energytransfer-causing combination of fluorescent materials (reporters).Furthermore, reagents were prepared in which each of these materialslabeled with the fluorescent materials, or each of the fluorescentmaterials themselves, was labeled with a leucine zipper peptide(materials for stabilizing the reporters in a spatially proximatestate). These reagents and a sample were mixed, an immunocomplex wasformed, and when one of the fluorescent materials was excited it wasfound that, due to binding based on the affinity among the materials,within the reaction complex, having weak affinity for each other, thefluorescent materials were brought in spatial proximity to each other,and a more efficient energy transfer occurred, and the present inventionwas thus completed.

[0017] That is, the present invention relates to a measurement methodfor a subject material (X) to be measured, the measurement method usinga first reagent comprising a material (A) that can bond to the subjectmaterial (X) to be measured, the material (A) being labeled with a firstreporter (R1), and a second reagent comprising a material (B) that canbond to the subject material (X) to be measured at a different site fromthat at which the material (A) bonds, the material (B) being labeledwith a second reporter (R2) that causes interaction with the firstreporter (R1),

[0018] wherein the first reagent includes a material (C) that bonds tothe first reporter (R1), the material (A) and the first reporter (R1)being directly bonded or bonded via the material (C) to form the reagent(A-R1-C or A-C-R1),

[0019] wherein the second reagent includes a material (D) that bonds tothe second reporter (R2) and has affinity for the material (C), thematerial (B) and the second reporter (R2) being directly bonded orbonded via the material (D) to form the reagent (B-R2-D or B-D-R2), and

[0020] wherein the first reagent, the second reagent and the subjectmaterial to be measured form a reaction complex, and due to bindingbased on the affinity in the reaction complex of the material (C) of thefirst reagent with the material (D) of the second reagent, the firstreporter (R1) and the second reporter (R2) are stabilized in a spatiallyproximate state, thereby causing a measurable interaction between thetwo reporters.

[0021] Moreover, the present invention relates to the above-mentionedmeasurement method wherein the subject material (X) to be measured is amaterial or a part thereof selected from the group consisting ofproteins, peptides, antigens, antibodies, lectins, lectin-bindingcarbohydrates, tumor markers, cytokines, cytokine receptors, hormones,hormone receptors, cell adhesion molecules, cell adhesion moleculeligands, nucleic acids, sugar chains, and lipids; a cell; anintracellular organelle; or a low molecular weight compound.

[0022] Furthermore, the present invention relates to the above-mentionedmeasurement method wherein the material (A) and/or the material (B) arematerials or parts thereof selected from the group consisting ofproteins, peptides, antigens, antibodies, lectins, lectin-bindingcarbohydrates, tumor markers, cytokines, cytokine receptors, hormones,hormone receptors, cell adhesion molecules, cell adhesion moleculeligands, nucleic acids, sugar chains, and lipids; or low molecularweight compounds.

[0023] Moreover, the present invention relates to the above-mentionedmeasurement method wherein the material (C) and/or the material (D) arematerials comprising one or two or more materials selected from thegroup consisting of proteins, peptides, nucleic acids, and sugar chains;or low molecular weight compounds.

[0024] Furthermore, the present invention relates to the above-mentionedmeasurement method wherein the first reporter (R1) and the secondreporter (R2) are different fluorescent materials, and the measurableinteraction between the reporters comprises a fluorescence energytransfer.

[0025] Moreover, the present invention relates to the above-mentionedmeasurement method wherein the first reporter (R1) is an enzyme thatcatalyzes bioluminescence, and the second reporter (R2) is an acceptorfor non-radiative energy transfer of the bioluminescence catalyzed bythe first reporter (R1).

[0026] Furthermore, the present invention relates to the above-mentionedmeasurement method wherein the first reporter (R1) is an enzyme thatcatalyzes chemiluminescence, and the second reporter (R2) is an acceptorfor non-radiative energy transfer of the chemiluminescence catalyzed bythe first reporter (R1).

[0027] Moreover, the present invention relates to the above-mentionedmeasurement method wherein the first reporter (R1) and the secondreporter (R2) are molecules that form parts of an enzyme, wherein eachof the first reporter (R1) and the second reporter (R2) individually hasdeleted or reduced enzyme activity, but the enzyme activity is generatedor increased by an interaction between the first reporter (R1) and thesecond reporter (R2).

[0028] Furthermore, the present invention relates to the above-mentionedmeasurement method wherein the material (A) and the material (B) areantibodies, antibody-derived single chain Fv, Fv, a part of Fv, Fab′, orFab, or mutants thereof, each thereof recognizing different sites in thesubject material (X) to be measured.

[0029] Moreover, the present invention relates to the above-mentionedmeasurement method wherein the material (A), the material (B), thereporter (R1), the reporter (R2), the material (C), and the material (D)comprise peptidergic materials, and the first reagent and the secondreagent comprise fusion proteins.

[0030] Furthermore, the present invention relates to the above-mentionedmeasurement method wherein the material (C) and the material (D) areboth leucine zipper peptides.

[0031] Moreover, the present invention relates to the above-mentionedmeasurement method wherein the measurement is carried out in ahomogenous system in which the first reagent and the second reagent aremade to act simultaneously on the subject material to be measured.

[0032] Furthermore, the present invention relates to the above-mentionedmeasurement method wherein the measurement is carried out in aheterogeneous system in which, among the first reagent and the secondreagent, one of the reagents is immobilized in advance on a solid phase,and then the other reagent and the subject material to be measured aremade to act on the immobilized reagent.

[0033] Moreover, the present invention relates to a measurement reagentfor use in the above-mentioned measurement method, the measurementreagent including the material (A), the material (B), the reporter (R1),the reporter (R2), the material (C), and the material (D).

[0034] Furthermore, the present invention relates to a measurement kitthat includes the above-mentioned measurement reagent.

[0035] ‘Measurement’ referred to here denotes measurement of the subjectmaterial to be measured by measurement of a signal emitted by thereporter, and includes simply measuring and detecting the presence ofthe subject material to be measured, for example, measuring thedistribution of the presence of the subject material to be measured incells or tissue, and measuring the concentration of the subject materialto be measured.

BRIEF DESCRIPTION OF DRAWINGS

[0036]FIG. 1 shows a schematic diagram of the measurement system of thepresent invention.

[0037]FIG. 2 shows a calibration curve for NP-labeled bovine albuminaccording to the measurement system of the present invention.

[0038]FIG. 3 shows a calibration curve for human albumin according tothe measurement system of the present invention.

MODES FOR CARRYING OUT THE INVENTION

[0039] The measurement method according to the present invention ischaracterized in that it uses a first reagent A-(R1-C) and a secondreagent B-(R2-D), wherein further introduced to each of A-R1, whichcomprises a material (A) labeled with a first reporter (R1), thematerial (A) having affinity for a subject material (X) to be measured;and B-R2, which comprises a material (B) labeled with a second reporter(R2), the material (B) having affinity for the subject material (X) tobe measured, are a material (C) and a material (D) having weak affinityfor each other. The material (A) and the material (B) are able to bindto different sites of the subject material (X) to be measured.

[0040] It is desirable for the bonding between each of the materials inthe first reagent A-(R1-C) to be such that the properties of eachmaterial are not lost or weakened by the other materials and, althoughthere is no limitation in the combination of these bonds, in order forthe reporters to be brought close together by the binding of thematerial (C) and the material (D) it is preferred that the combinationis A-R1-C or A-C-R1. The same applies to the second reagent B-(R2-D).

[0041] A notation such as A-(R1-C) is conveniently used as the notationfor the reagent, but the intention is to show that either of the twoparts inside the parentheses are bonded to the material outside theparentheses, and unless otherwise stated represents A-R1-C and A-C-R1.

[0042] A complex (C-R1)-A:X:B-(R2-D) is formed by mixing the firstreagent (C-R1)-A and the second reagent (D-R2)-B with a sample in whichthe measured material X is present and, moreover, as a result of theeffect of increasing the local density within the complex, the material(C) and the material (D) bind together to give a more stable complex.Furthermore, even though the first reagent (C-R1)-A and the secondreagent (D-R2)-B are in a bound state by virtue of the weak interactionbetween C and D, binding of the subject material X to be measured withthe first reagent (C-R1)-A and the second reagent (D-R2)-B forms astable complex.

[0043] Moreover, a stable complex is also formed in the same way in thecase where the binding of the subject material X to be measured with thefirst reagent A-(R1-C) and the second reagent B-(R2-D), and the binding(C:D) between the material (C) and the material (D) occursimultaneously. As a result, the proximate state of R1 and R2 in thecomplex is stabilized, and a more efficient energy transfer is observed.On the other hand, when the subject material X to be measured is notpresent, the complex (C-R1)-A:X:B-(R2-D) is not formed, and since theinteraction of C with D is weak, even if a complex A-(R1-C):(D-R2)-B isformed it is extremely unstable, and efficient energy transfer is notobserved.

[0044] The subject material X to be measured in the measurement methodof the present invention is not particularly limited as long as it is amaterial in which a material with binding capacity for the abovematerials is present.

[0045] With regard to this kind of subject material X to be measured, asexamples thereof there can be cited peptidergic materials such asproteins and peptides, antigens, antibodies, lectins, lectin-bindingcarbohydrates, tumor markers, cytokines, cytokine receptors, hormones,hormone receptors, cell adhesion molecules, cell adhesion moleculeligands, nucleic acids, sugar chains, lipids, etc., or a part thereof,cells, intracellular organelles, low molecular weight compoundsgenerally called haptens, etc.

[0046] The materials A and B that have binding capacity for the subjectmaterial to be measured in the measurement method of the presentinvention are not particularly limited as long as they are materialsthat bind specifically to the subject material to be measured, but it isnecessary for them to have no affinity for each other and for each ofthem to recognize and bind to different parts of the subject material Xto be measured. For example, in the case where the subject material tobe measured is an antigen, the materials A and B could be antibodiesthat recognize separate epitopes. With regard to the antibodies, notonly IgG obtained by the immunization of animals, but also antibodyfragments such as the IgG papain digestion fragment Fab and the pepsindigestion fragment F(ab′)₂; or Fv fragment, scFv (single chain Fv),etc., obtained by genetic engineering can be used.

[0047] With regard to the materials A and B, peptidergic materials suchas proteins and peptides, antibodies, antigens, lectins, lectin-bindingcarbohydrates, tumor markers, cytokines, cytokine receptors, hormones,hormone receptors, cell adhesion molecules, cell adhesion moleculeligands, nucleic acids, sugar chains, and lipids, or a part thereof, orlow molecular weight compounds such as haptens, etc., can also be used.Moreover, in the case where the subject material X to be measured is anantibody, a hapten that can be recognized by X can be employed as thematerial A, and a material that can bind to a different site of X fromthat where material A binds can be employed as the material B.

[0048] The first reporter (R1) and the second reporter (R2) are used asa set of reporters, and generate a measurable signal by interactiontherebetween. Measurement of the subject material X to be measured inthe measurement method of the present invention is carried out bymeasuring the signal by an appropriate measurement method.

[0049] The reporters R1 and R2 in the measurement method of the presentinvention are not particularly limited as long as, in the case of FRET,the combination thereof results in fluorescence energy transfer, butwith regard to R1, fluorescent proteins such as GFP (Clontech) and GFPmutants, fluorescent materials such as fluorescein, etc., can beconsidered, and with regard to R2, fluorescent proteins such as GFP andGFP mutants, fluorescent materials such as rhodamine, etc., can beconsidered.

[0050] Furthermore, in the case of BRET they are not particularlylimited as long as the combination thereof results inbioluminescence-fluorescence energy transfer. With regard to thereporter R1, enzymes catalyzing bioluminescence or modifications thereofcan be used, and examples thereof include luminescent enzymes etc.derived from luminescent bacteria, fireflies, sea fireflies, Pyrophorusnoctilucus, and Renilla reniformis. Furthermore, R1 may also be anenzyme that catalyzes chemiluminescence or a modification thereof, and aperoxidase can be cited as an example thereof. With regard to aluminescent enzyme substrate, in the case of a firefly-derivedluciferase, luciferin can be cited, and in the case of a peroxidase,luminol can be cited. With regard to examples of the reporter R2, therecan be cited fluorescent proteins such as GFP and GFP mutants,fluorescent materials such as rhodamine, etc.

[0051] In addition, a combination of R1 and R2 can be used wherein R1and R2 are molecules that form parts of an enzyme and each individuallyhas deleted or reduced enzyme activity, but wherein an interactionbetween R1 and R2 generates or increases the enzyme activity. Asexamples of this kind of reporter there can be cited the β-galactosidaseΔα mutant and Δω mutant, etc.

[0052] In the present invention, the distance between the reporters R1and R2 at which a measurable signal is generated is preferablystabilized at 0 to 50 nm, and particularly 10 nm or below.

[0053] The material C and the material D in the measurement method ofthe present invention are not particularly limited as long as they arematerials having weak affinity for each other, and peptidergic materialssuch as proteins and peptides, nucleic acids, sugar chains, etc., orcomplexes thereof can be used. Specifically, for example, peptidefragments such as leucine zipper sequence (leucine zipper peptide)occurring in eukaryotic nuclear transcription factors such as c-Jun andc-Fos can be prepared by genetic engineering and used. In this case,mutations can be introduced into these amino acid sequences, therebyregulating the strength of the affinity. Furthermore, DNA sequences,etc. capable of complementing one another can be used. In this case,regulating the length and GC content of these DNA sequences can alsoregulate the strength of the affinity. Moreover, as the material C, alow molecular weight compound generally called a hapten can be employed,and as the material D an antibody or an antibody fragment that canrecognize the material C can be used.

[0054] With regard to the preparation of the first reagent A-(R1-C) andthe second reagent B-(R2-D) in the measurement method of the presentinvention, in the case where the material A (B), the reporter R1 (R2),and the material C (D) are peptidergic materials, the preparation of thereagent A-(R1-C) (B-(R2-D)) as a genetically engineered fusion proteinis preferred from the point of view of the possibility of site-specificlabeling and shortening the manufacturing process, but a preparationinvolving preparing each of the material A (B), reporter R1 (R2), andmaterial C (D) and chemically bonding each one is also possible.Furthermore, antibody fragments can be labeled with R1 (R2), C (D), etc.via proteins such as protein G and protein A having affinity forantibody fragments, and the reagent A-(R1-C) (B-(R2-D)) can also beprepared employing an avidin-biotin interaction.

[0055] Since the measurement method of the present invention employsfluorescence energy transfer and luminescence-fluorescence energytransfer, separation of unreacted reagents A-(R1-C), B-(R2-D) isunnecessary, the washing operation that is carried out in the usualsandwich method does not have to be carried out, and since theconcentration of a subject material to be measured can be measured,measurement is possible in both a homogeneous system and a heterogeneoussystem, but for convenience measurement in a homogeneous system isdesirable.

[0056] In the case of measurement of a heterogeneous system, one of thereagents A-(R1-C) and B-(R2-D) is immobilized on a solid phase, mixedwith the other reagent and a sample, and reacted for a fixed time, andmeasurement can then be carried out as it is or after carrying out awashing operation. With regard to the solid phase, materials such aslatex particles, polystyrene beads, and ELISA microplates, which aresuitably used in normal immunoassays, can be used.

[0057] The measurement system of the present invention will now bespecifically explained by reference to a schematic diagram (FIG. 1), butthe present invention is not limited thereto.

[0058] In the case where measurement is carried out on an antigen (X) asthe subject material to be measured, two antibody fragments (1) and (2)(A, B), which bind to different sites on the antigen, are prepared. Afluorescent protein (1) (the fluorescence energy donor R1) is bonded tothe antibody fragment (1), and a leucine zipper (C) is further bonded.On the other hand, a fluorescent protein (2) (the fluorescence energyacceptor R2) is bonded to the antibody fragment (2), and a leucinezipper (D) is further bonded.

[0059] Therefore, the two types of reagent (A-R1-C), which includes theantibody fragment (1), the fluorescent protein (1), and the leucinezipper, and (B-R2-D), which includes the antibody fragment (2), thefluorescent protein (2), and the leucine zipper, are used in themeasurement system of FIG. 1.

[0060] By adding the antigen (X), which is the subject material to bemeasured, to these two types of reagent so that they are all presenttogether, as shown in FIG. 1, a reaction complex ((C-R1)-A:X:B-(R2-D))is formed.

[0061] When the reaction complex is formed, the fluorescent protein (1)(R1) and the fluorescent protein (2) (R2) are arranged in proximity toone another by the binding of the two leucine zippers (C, D) as a resultof them affinity binding together. In FIG. 1, because the reporters arefluorescent proteins, the object of the present invention can beachieved without the necessity for binding as long as they are inproximity to one another but, according to the type of reporter, theymay be also bound as necessary. In the case where the measurement systemis a homogenous system, in order that a non-specific signal is notgenerated, it is preferable for the binding between the reporters to beweak or for there to be no binding.

[0062] As shown in FIG. 1, when the fluorescence resonance energy donor(R1) alone is irradiated with excitation light λ(1), fluorescence λ(1)′is generated. When a reaction complex such as that shown in FIG. 1 isformed, the fluorescence resonance energy donor (R1) and thefluorescence resonance energy acceptor (R2) are in proximity to oneanother, and fluorescence energy transfer occurs between them.Therefore, when the reaction complex is irradiated with the excitationlight λ(1) fluorescence energy transfer occurs, and as a resultfluorescence λ(2)′ is generated.

[0063] Since the efficiency of the fluorescence energy transfer isinversely proportional to the 6th power of the distance between R1 andR2, from the point of view of a stronger fluorescence being obtained inthe λ(2)′ generated as a result of C and D being brought into proximityto each other than is the case where they are not brought into proximityto each other, it is suitable for high sensitivity measurement and,moreover, since R1 and R2 are always arranged at a fixed distance in thereaction complex by C and D, it is excellent from the point of obtaininga stable signal.

[0064] In this way, in the case where a fluorescent protein is used asthe reporter, the fluorescence by fluorescence energy transfer gives ameasurable signal, and by measuring this fluorescence, detection andconcentration measurement of the subject material to be measured becomepossible.

[0065] The subject material to be measured is detected by measuringchanges in the fluorescence spectrum attributable to fluorescence energytransfer, or changes in the fluorescence intensity ratio of λ(1)′ andλ(2)′.

[0066] In the case where the concentration of the subject material to bemeasured is measured, a calibration curve of the fluorescence intensityratio of λ(1)′ and λ(2)′ as an index is created using standardmaterials, and measurement is carried out by applying the fluorescenceintensity ratio of λ(1)′ and λ(2)′ of the subject material to bemeasured to the calibration curve.

[0067] The present invention is explained further in detail below bymeans of examples, but the present invention is not limited thereby.

EXAMPLE 1 Isolation of Fluorescent Protein EYFP and ECFP DNA

[0068] For EYFP, EGFP DNA was amplified by means of PCR using Pfu DNApolymerase (Stratagene) with pEGFP (Clontech) as a template. The primers

[0069] 5′-CCGCGGCCGCCATGGTGAGCAAGGGCGAGGAGCTG-3′ (SEQ ID No. 1) and

[0070] 5′-CCCTCGAGCTTGTACAGCTCGTCCATGCCGAG-3′ (SEQ ID No. 2) were used.The product of PCR was digested with the restriction enzymes NotI andXhoI, then separated and purified by 1% agarose gel electrophoresis, andinserted into the NotI and XhoI sites of pBluescript II KS+ (Stratagene)(pBS/EGFP). Based on this pBS/EGFP and using the following two primers

[0071] 5′-CCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACC-3′ (SEQID No. 3) and

[0072] 5′-CCACTACCTGAGCTACCAGTCCGCCCTGAG-3′ (SEQ ID No. 4) site-directedmutagenesis was carried out by means of the Kunkel method (the 4residues substituted were S65G, V68L, S72A, and T203Y) to give EYFP.

[0073] For ECFP, ECFP DNA was amplified by means of PCR using Pfu DNApolymerase with pECFP-C1 (Clontech) as a template. The primers

[0074] 5′-CCGCGGCCGCCATGGTGAGCAAGGGCGAGGAGCTG-3′ (SEQ ID No. 5) and

[0075] 5′-CCCTCGAGCTTGTACAGCTCGTCCATGCCGAG-3′ (SEQ ID No. 6) were used.

[0076] The EYFP DNA and ECFP DNA fragments so prepared were digested bythe restriction enzymes NotI and XhoI, and subjected to 1% agarose gelelectrophoresis, thereby cutting out and purifying each fragment.

EXAMPLE 2 Preparation of Flexible Linker DNA

[0077] Using the two primers below and Pfu DNA polymerase, by carryingout an annealing/growth reaction a flexible linker (called ‘FL4’ below)DNA fragment was prepared.

[0078] The primers

[0079] 5′-CCCAAGCTTTCCGGCGGGGGTGGCTCCGGCGGGGGTGGATCCGGTGGCGGTGGCTC-3′(SEQ ID No. 7) and

[0080] 5′-CCCCGCGGCCGCGCTACCGCCACCGCCGGAGCCACCGCCACCGGAT-3′ (SEQ ID No.8) were used.

[0081] The amino acid sequence of the flexible linker so prepared was

[0082] GGGGSGGGGSGGGGSGGGGS (SEQ ID No. 9)

[0083] The flexible linker DNA fragment so prepared was digested withthe restriction enzymes HindIII and NotI, and subjected to 2% agarosegel electrophoresis, thereby cutting out and purifying the fragment.

EXAMPLE 3 Cloning of Leucine Zipper Sequence from c-Jun and FosB GeneProducts

[0084]E. coli HB101 containing the c-Jun and FosB gene products pJac-1and pBSKS-FosB (Riken DNA Bank) was cultured overnight at 37° C. in 5 mLof LB broth (50 μg/mL ampicillin), and the plasmid DNA was purified bythe alkaline-SDS method. Leucine zipper (called ‘Lzip’ below) DNA ofeach of the c-Jun and FosB was amplified by means of PCR using Pfu DNApolymerase with the purified plasmid DNA as a template. As primers,those below were used.

[0085] For c-Jun amplification

[0086] 5′-CCCCGGATCCGTCGACGAATTCAGTGGTTCATGACTTTCTGCTTAAGCTGTG-3′ (SEQID No. 10),

[0087] 5′-CCCCCCTCGAGGGTGGCCGGATCGCTCGGCTAGAGG-3′ (sequence No. 11);

[0088] For FosB amplification

[0089] 5′-CCCCGGATCCGTCGACGAATTCAGTGGGCCACCAGGACAAACTC-3′ (SEQ ID No.12)

[0090] 5′-CCCCCCTCGAGGGTGGCCTGACAGATCGACTTCAGGCGG-3′ (SEQ ID No. 13)

[0091] The PCR reaction liquid was purified by phenol-chloroformextraction and ethanol precipitation, and restriction enzyme digestedusing SalI (Takara Shuzo Co., Ltd.) and XhoI (Takara Shuzo Co., Ltd.).The restriction enzyme digestion preparations were subjected to 1%agarose gel electrophoresis, and fragments of approximately 160 bp eachwere cut out and purified.

[0092] It was confirmed that a base sequence determined for the clonedLzip DNA coincided with the leucine zipper sequence described in theliterature.

EXAMPLE 4 Preparation of Anti-NP (4-Hydroxy-3-Nitrophenyl-Acetyl)Antibody Expression Vector

[0093] pET TRX Fusion System 32 (Novagen) was used as the fusion proteinexpression system.

[0094] Anti-NP (4-hydroxy-3-nitrophenyl-acetyl; abbreviated to ‘NP’below) antibody ScFv DNA fragment was isolated by restriction enzymeEcoRV and HindIII digestion from pScFv(NP)AP 5.9 kbp vector (J.Biochem., 122, 322-329 (1997)). This anti-NP antibody ScFv DNA fragmentwas inserted into EcoRV and HindIII sites of pET32 vector (Novagen)(pET32/Trx-ScFv(NP)). The FL4 DNA fragment prepared in Example 2 wasthen inserted into the HindIII and NotI sites of the pET32/Trx-ScFv(NP)(pET32/Trx-ScFv(NP)-FL4).

[0095] Next, the EYFP DNA fragment and ECFP DNA fragment prepared inExample 1 were inserted into the NotI and XhoI sites of thepET32/Trx-ScFv(NP)-FL4 (pET32/Trx-ScFv(NP)-FL4-EYFP,pET32/Trx-ScFv(NP)-FL4-ECFP). Moreover, the c-Jun and FosB Lzip DNAfragments prepared in Example 3 were inserted into the XhoI sites of thepET32/Trx-ScFv(NP)-FL4-EYFP and pET32/Trx-ScFv(NP)-FL4-ECFP. The sizeand direction of the inserted fragments in the vectors so prepared wereconfirmed by restriction enzyme digestion.

[0096] The anti-NP antibody expression vectors were the four types ofexpression vector below.

[0097] (1) pET32/Trx-ScFv(anti-NP)-FL4-EYFP-Lzip(c-Jun)

[0098] (2) pET32/Trx-ScFv(anti-NP)-FL4-ECFP-Lzip(FosB)

[0099] (3) pET32/Trx-ScFv(anti-NP)-FL4-EYFP

[0100] (4) pET32/Trx-ScFv(anti-NP)-FL4-ECFP

EXAMPLE 5 Preparation of NP-Immobilized Sepharose 4B Column

[0101] To 3 mL of EAH-Sepharose 4B (Pharmacia) was added 21 mg ofNP-CAP-Osu (CRB) (dissolved in 100 μL of DMF), and it was stirred gentlyovernight at 4° C. The following day, the NP-immobilized Sepharose 4Bwas packed in a disposable column, the gel was washed with 30 mL of 0.1M Tris buffer/100 mM NaCl (pH 7.5), then repeatedly washed alternatelywith 6 mL of 0.1 M acetate buffer/0.1 M NaCl (pH 4.0), 6 mL of 50 mMTris buffer/50 mM NaCl (pH 8.0), 6 mL of 0.1 M acetate buffer/0.1 M NaCl(pH 4.0), 6 mL of 50 mM Tris buffer/50 mM NaCl (pH 8.0), 6 mL of 0.1 Macetate buffer/0.1 M NaCl (pH 4.0), and 6 mL of 50 mM Tris buffer/50 mMNaCl (pH 8.0), and finally washed with 9 mL of 0.1 M Tris buffer/0.1 MNaCl (pH 7.5).

EXAMPLE 6 Expression and Purification of Anti-NP Antibody Fusion Protein

[0102] Transformation of E. Coli Origami (DE3) (Novagen) was carried outusing the anti-NP antibody expression vectors (1), (2), (3), and (4).The transformed clones were cultured overnight at 30° C. with shaking in5 mL of LB broth (50 μg/mL ampicillin, 15 μg/mL kanamycin, 12.5 μg/mLtetracycline). The following day, the cultured cells were subcultured in1.5 L of LB broth (50 μg/mL ampicillin, 15 μg/mL kanamycin, 12.5 μg/mLtetracycline: 5 L baffled flask) and culturing was continued at 30° C.When the turbidity (O.D. 600) of the culture liquid reachedapproximately 0.5 (approximately 6 hours of culturing), 1.5 mL of 0.1 MIPTG was added, the temperature was lowered to 16° C., and culturing wascontinued overnight. The following day, the cells were collected bycentrifugation, 40 mL of 0.1 M phosphate buffer (pH 7.2) was added toeach sample, and the cells were suspended. These cell liquids were thenput through two repetitions of being kept at −80° C. and at roomtemperature while being shielded from light. After carrying out thefreeze-thawing an ultrasonic treatment was carried out to furtherdisrupt the cells.

[0103] The ultrasonically-treated samples were centrifuged, and thesupernatants were collected. To each of the collected supernatants wasadded 500 μL of the NP-immobilized Sepharose 4B (Example 5), and theywere gently stirred overnight at 4° C. while being shielded from light.The following day, the gels were packed in disposable column containers,and washed with a flow of 10 mL of 0.1 M Tris buffer/0.1 M NaCl (pH7.5). Following this, 5 mL of 0.5 mg/mL NP solution (one in which it hadbeen dissolved in 1 M Tris buffer (pH 8.0)) was added, and elution ofthe target protein was carried out. The eluate was collected 1 mL at atime in Eppendorf tubes. The collected samples were dialyzed against 0.1M phosphate buffer (pH 7.2).

EXAMPLE 7 Preparation of NP-Labeled Bovine Albumin

[0104] To 50 mg of bovine albumin (Sigma) dissolved in 1 mL of 0.1 Msodium bicarbonate buffer (pH 8.0) was added 14 mg of NP-CAP-Osu (CRB)(dissolved in 100 μL of DMF), and the mixture was gently stirredovernight (4° C.) while being shielded from light. The following day,the reaction liquid was put on a PD-10 column (Pharmacia) and elutedwith 0.1 M phosphate buffer (pH 7.2). Since the NP is yellow, separationof the NP-labeled bovine albumin and unreacted NP-CAP-Osu on the columncould be confirmed, the earlier-eluting peak was fractionated, and thiswas the NP-labeled bovine serum albumin. The O.D. 280 and O.D. 420 ofthe fractionated NP-labeled bovine serum albumin were measured by aspectrophotometer (Shimadzu Corporation), and when the NP labeling ratioof the bovine albumin was determined, the labeling ratio was NP/bovinealbumin=approximately 15.

EXAMPLE 8 Measurement of NP-Labeled Bovine Albumin Using Anti-NPAntibody/Fluorescent Protein/Leucine Zipper Fusion Protein

[0105] Measurement of the concentration of the NP-labeled bovine serumalbumin was carried out using anti-NP antibody/fluorescentprotein/leucine zipper fusion protein that had been expressed by E. ColiOrigami and purified on an NP-labeled Sepharose 4B column. Anti-NPantibody fusion proteins (Example 6) that had been affinity purified onan NP column were mixed in the combinationsTrx-ScFv(anti-NP)-FL4-EYFP-Lzip(c-Jun)/Trx-ScFv(anti-NP)-FL4-ECFP-Lzip(FosB),and Trx-ScFv(anti-NP)-FL4-EYFP/Trx-ScFv(anti-NP)-FL4-ECFP, so that theconcentration of each fusion antibody became 3 μg/mL, and 200 μL of eachwas injected into 1.5 mL Eppendorf tubes. 10 μL of NP-labeled bovinealbumin (Example 7) (at bovine albumin concentrations of 0, 10, and 100μg/mL; blank was 0.1 M phosphate buffer) was added thereto. They werethen allowed to stand for one hour at room temperature while beingshielded from light. After one hour, each sample was injected into thecell of a spectrofluorometer, and the fluorescence spectrum wasmeasured.

[0106] The fluorescence spectrum was measured (room temperature) from450 to 600 nm using a Hitachi F2000 spectrofluorometer (Hitachi Ltd.)and exciting at 433 nm. It was found that an NP-labeled bovine albuminconcentration-dependent change in the fluorescence spectrum was onlyobserved in the case where a fusion protein into which leucine zipperhad been introduced was used. When a calibration curve of addedNP-labeled bovine albumin was prepared with the EYFP and ECFPfluorescence intensity ratio as the fluorescence energy transferefficiency, as shown in FIG. 2, by introducing leucine zipper it couldbe confirmed that the FRET efficiency was enhanced and the measurementsensitivity was improved.

EXAMPLE 9 Extraction of Anti-Human Albumin Antibody Nos. 11 and 13 mRNAand Synthesis of cDNA)

[0107] Culturing of a hybridoma (home-grown) secreting anti-humanalbumin monoclonal antibodies No. 11 and No. 13 was carried out usingRPMI culture medium (Sigma) containing 10% fetal bovine serum. When thecell count reached approximately 10⁷ the cultured cells were collectedby centrifugation. Extraction of mRNA from the collected hybridoma wascarried out using a QuickPrep Micro mRNA Purification Kit (Pharmacia).Synthesis of cDNA was carried out using a First-Strand cDNA SynthesisKit (Pharmacia) with 2.0 μg of the mRNA so obtained as a template.

EXAMPLE 10 Preparation of Anti-Human Albumin Antibody Nos. 11 and 13Single Chain Fv)

[0108] Preparation of anti-human albumin monoclonal antibody Nos. 11 and13 single chain Fv DNA was carried out according to the known methoddescribed in ‘Antibody Engineering’ (Oxford Press) with the anti-humanalbumin monoclonal antibody No. 11 and No. 13 cDNA (Example 9) as atemplate.

EXAMPLE 11 Preparation of Anti-Human Albumin Antibody Expression Vector

[0109] Preparation of anti-human albumin expression vector was carriedout by insertion of anti-human albumin antibody Nos. 11 and 13 ScFv(Example 10), cloned from the hybridoma-secreted anti-human albuminmonoclonal antibody, into anti-NP antibody ScFv fragments cut out fromanti-NP antibody expression vectors (1) to (4) (Example 4) byrestriction enzyme digestion.

[0110] PCR was carried out using primers into which restriction enzymesites (EcoRV, HindIII) had been introduced, with anti-human albuminantibody ScFv as a template. Restriction enzyme digestion (EcoRV,HindIII) of the product of the PCR was then carried out, it wassubjected to 1% agarose gel electrophoresis, and the anti-human albuminantibody ScFv fragments were cut out and purified. On the other hand,restriction enzyme digestion (EcoRV, HindIII: Takara Shuzo Co., Ltd.) ofpET32/Trx-ScFv(anti-NP)-FL4-EYFP-Lzip(c-Jun),pET32/Trx-ScFv(anti-NP)-FL4-ECFP-Lzip(FosB),pET32/Trx-ScFv(anti-NP)-FL4-EYFP, and pET32/Trx-ScFv(anti-NP)-FL4-ECFPprepared in Example 4 was carried out, they were subjected to 1% agarosegel electrophoresis, and vector parts other than anti-NP antibody ScFvfragments were cut out and purified. Anti-human albumin antibodyexpression vectors were prepared by inserting EcoRV and HindIII digestedNo. 11 and No. 13 antibody ScFv into the EcoRV and HindIII sites ofthese vectors. The size and direction of the inserted fragments in thevectors so prepared were confirmed by restriction enzyme digestion.

[0111] The anti-human albumin antibody expression vectors were the fourtypes of expression vector below.

[0112] A) pET32/Trx-ScFv(No.13)-FL4-EYFP-Lzip(c-Jun)

[0113] B) pET32/Trx-ScFv(No.11)-FL4-ECFP-Lzip(FosB)

[0114] C) pET32/Trx-ScFv(No.13)-FL4-EYFP

[0115] D) pET32/Trx-ScFv(No.11)-FL4-ECFP

EXAMPLE 12 Preparation of Human Albumin-Immobilized Sepharose 4B Column

[0116] 6 g of CNBr-Sepharose4B (Pharmacia) was weighed out and washedwith 1 mM hydrochloric acid (1.2 L). CNBr-Sepharose4B and human albumin(Sigma) were then mixed (0.1 M NaHCO₃, 0.5M NaCl pH 8.3) so that thehuman albumin concentration became 4 mg/mL, and gently stirred overnightat 4° C. The following day the human albumin-immobilized Sepharose 4Bwas washed alternately with 0.1 M acetate buffer pH 4.0 and 0.1 M Trisbuffer pH 8.0.

EXAMPLE 13 Expression and Purification of Anti-Human Albumin AntibodyFusion Protein

[0117] Transformation of E. Coli OrigamiB(DE3)pLysS (Novagen) wascarried out using the anti-human albumin antibody expression vectors A,B, C, and D (Example 11). The transformed E. Coli was cultured in 5 mLLB broth (50 μg/mL ampicillin, 15 μg/mL kanamycin, 12.5 μg/mLtetracycline, 34 μg/mL chloramphenicol) at 30° C. overnight withshaking. The following day, the cultured cells were subcultured in 1.5 Lof LB broth, and culturing was continued at 30° C. When the turbidity(O.D. 600) of the culture liquid reached approximately 0.5(approximately 6 hours of culturing), 1.5 mL of 0.1 M IPTG was added,the temperature was lowered to 16° C., and culturing was continuedovernight.

[0118] The following day, the cells were collected by centrifugation, 40mL of 0.1 M phosphate buffer (pH 7.2) was added to each sample, and thecells were suspended. These cell liquids were then put through tworepetitions of being kept at −80° C. and at room temperature while beingshielded from light. After carrying out the freeze-thawing an ultrasonictreatment was carried out to further disrupt the cells.

[0119] The ultrasonically-treated samples were centrifuged, and thesupernatants were collected. To each of the collected supernatants wasadded 500 μL of the human albumin-immobilized Sepharose 4B (Ref. Example12), and they were gently stirred overnight at 4° C. while beingshielded from light. The following day, the gels were packed in adisposable column, and washed with a flow of 20 mL of 0.1 M Trisbuffer/0.5 M NaCl (pH 8.0). 2.5 mL of 0.1 M Na₂HPO₄ NaOH (pH 12) wasthen added, and elution of the target protein was carried out. Theeluate was collected 500 μL at a time in Eppendorf tubes. To each of theeluate fractions was added 100 μL of 1 M Tris buffer (pH 6.5), andneutralization was effected.

EXAMPLE 14 Measurement of Human Serum Albumin Using Anti-Human AlbuminAntibody/Fluorescent Protein/Leucine Zipper Fusion Protein

[0120] Measurement of human albumin concentration was carried out usinganti-human albumin antibody/fluorescent protein/leucine zipper fusionprotein that had been expressed by E. Coli OrigamiB(DE3)pLysS andpurified with a human albumin-labeled Sepharose 4B column. Anti-humanalbumin antibody fusion proteins (Example 13) affinity purified with ahuman albumin-labeled column, in the combinationsTrx-ScFv(No.11)-FL4-ECFP-Lzip(FosB)/Trx-ScFv(No.13)-FL4-EYFP-Lzip(c-Jun),and Trx-ScFv(No.11)-FL4-ECFP/Trx-ScFv(No.13)-FL4-EYFP, were mixed with50 mM Tris buffer/50 mM NaCl/0.1% gelatin (pH 8.0) so that theconcentration of each fusion antibody became 3 μg/mL, and 190 μL of eachwas injected into a 1.5 mL Eppendorf tube. 10 μL of human albumin(Sigma) (0, 3.1, 6.3, 12.5, 25, 50, and 100 μg/mL; diluted with 50 mMTris buffer/50 mM NaCl/0.1% gelatin (pH 8.0)) was added thereto. Theywere then allowed to stand for one hour at room temperature while beingshielded from light. After one hour, each sample was injected into thecell of a spectrofluorometer, and the fluorescence spectrum wasmeasured. The fluorescence spectrum was measured (room temperature) from450 to 600 nm using a Hitachi F2000 spectrofluorometer and exciting at433 nm.

[0121] In the same way as with the results for NP-labeled bovinealbumin, it was found that a human albumin concentration-dependentchange in the fluorescence spectrum could only be observed in the casewhere a leucine zipper was introduced. When a calibration curve of humanalbumin was prepared with the EYFP and ECFP fluorescence intensity ratioas the fluorescence energy transfer efficiency, as shown in FIG. 3, aswas the case where NP-labeled bovine albumin was used, with acombination of two types of monoclonal antibody, by introducing leucinezipper it could also be confirmed that the FRET efficiency was increasedand the sensitivity of measurement was improved.

Industrial Applicability

[0122] In accordance with the measurement method of the presentinvention, efficient energy transfer can be reliably obtained, and theconcentration of a material can be measured with a low background, highprecision and high sensitivity. Furthermore, measurement of ahomogeneous system is also possible, and in particular since a washingoperation is not necessary, it is also simple to use in a clinicalenvironment.

1 13 1 35 DNA Artificial Sequence Description of ArtificialSequencePrimer for amplifing EGFP DNA by PCR 1 ccgcggccgc catggtgagcaagggcgagg agctg 35 2 32 DNA Artificial Sequence Description ofArtificial SequencePrimer for amplifing EGFP DNA by PCR 2 ccctcgagcttgtacagctc gtccatgccg ag 32 3 54 DNA Artificial Sequence Description ofArtificial SequencePrimer for site-directed mutagenesis by Kunkel method3 ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc gacc 54 4 30DNA Artificial Sequence Description of Artificial SequencePrimer forsite-directed mutagenesis by Kunkel method 4 ccactacctg agctaccagtccgccctgag 30 5 35 DNA Artificial Sequence Description of ArtificialSequencePrimer for amplifing ECFP DNA by PCR 5 ccgcggccgc catggtgagcaagggcgagg agctg 35 6 32 DNA Artificial Sequence Description ofArtificial SequencePrimer for amplifing ECFP DNA by PCR 6 ccctcgagcttgtacagctc gtccatgccg ag 32 7 56 DNA Artificial Sequence Description ofArtificial SequencePrimer for producing flexible linker DNA 7 cccaagctttccggcggggg tggctccggc gggggtggat ccggtggcgg tggctc 56 8 46 DNAArtificial Sequence Description of Artificial SequencePrimer forproducing flexible linker DNA 8 ccccgcggcc gcgctaccgc caccgccggagccaccgcca ccggat 46 9 20 PRT Artificial Sequence Description ofArtificial SequenceFlexible Linker 9 Gly Gly Gly Gly Ser Gly Gly Gly GlySer Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 10 52 DNAArtificial Sequence Description of Artificial SequencePrimer foramplifing c-Jun by PCR 10 ccccggatcc gtcgacgaat tcagtggttc atgactttctgcttaagctg tg 52 11 36 DNA Artificial Sequence Description of ArtificialSequencePrimer for amplifing c-Jun by PCT 11 cccccctcga gggtggccggatcgctcggc tagagg 36 12 44 DNA Artificial Sequence Description ofArtificial SequencePrimer for amplifing FosB by PCR 12 ccccggatccgtcgacgaat tcagtgggcc accaggacaa actc 44 13 39 DNA Artificial SequenceDescription of Artificial SequencePrimer for amplifing FosB by PCR 13cccccctcga gggtggcctg acagatcgac ttcaggcgg 39

1. A measurement method for a subject material (X) to be measured, themeasurement method using a first reagent comprising a material (A) thatcan bond to the subject material (X) to be measured, the material (A)being labeled with a first reporter (R1), and a second reagentcomprising a material (B) that can bond to the subject material (X) tobe measured at a different site from that at which the material (A)bonds, the material (B) being labeled with a second reporter (R2) thatcauses interaction with the first reporter (R1), wherein the firstreagent includes a material (C) that bonds to the first reporter (R1),the material (A) and the first reporter (R1) being directly bonded orbonded via the material (C) to form the reagent (A-R1-C or A-C-R1),wherein the second reagent includes a material (D) that bonds to thesecond reporter (R2) and has affinity for the material (C), the material(B) and the second reporter (R2) being directly bonded or bonded via thematerial (D) to form the reagent (B-R2-D or B-D-R2), wherein the firstreagent, the second reagent, and the subject material to be measuredform a reaction complex, and wherein due to binding based on theaffinity in the reaction complex of the material (C) of the firstreagent with the material (D) of the second reagent, the first reporter(R1) and the second reporter (R2) are stabilized in a spatiallyproximate state, thereby causing a measurable interaction between thetwo reporters.
 2. The measurement method according to claim 1 whereinthe subject material (X) to be measured is a material or a part thereofselected from the group consisting of proteins, peptides, antigens,antibodies, lectins, lectin-binding carbohydrates, tumor markers,cytokines, cytokine receptors, hormones, hormone receptors, celladhesion molecules, cell adhesion molecule ligands, nucleic acids, sugarchains, and lipids; a cell; an intracellular organelle; or a lowmolecular weight compound.
 3. The measurement method according to claim1 or 2 wherein the material (A) and/or the material (B) are materials orparts thereof selected from the group consisting of proteins, peptides,antigens, antibodies, lectins, lectin-binding carbohydrates, tumormarkers, cytokines, cytokine receptors, hormones, hormone receptors,cell adhesion molecules, cell adhesion molecule ligands, nucleic acids,sugar chains, and lipids; or low molecular weight compounds.
 4. Themeasurement method according to any of claims 1 to 3 wherein thematerial (C) and/or the material (D) are materials comprising one or twoor more materials selected from the group consisting of proteins,peptides, nucleic acids, and sugar chains; or low molecular weightcompounds.
 5. The measurement method according to any of claims 1 to 4wherein the first reporter (R1) and the second reporter (R2) aredifferent fluorescent materials, and the measurable interaction betweenthe reporters comprises a fluorescence energy transfer.
 6. Themeasurement method according to any of claims 1 to 4 wherein the firstreporter (R1) is an enzyme that catalyzes bioluminescence, and thesecond reporter (R2) is an acceptor for non-radiative energy transfer ofthe bioluminescence catalyzed by the first reporter (R1).
 7. Themeasurement method according to any of claims 1 to 4 wherein the firstreporter (R1) is an enzyme that catalyzes chemiluminescence, and thesecond reporter (R2) is an acceptor for non-radiative energy transfer ofthe chemiluminescence catalyzed by the first reporter (R1).
 8. Themeasurement method according to any of claims 1 to 4 wherein the firstreporter (R1) and the second reporter (R2) are molecules that form partsof an enzyme, wherein each of the first reporter (R1) and the secondreporter (R2) individually has deleted or reduced enzyme activity, butthe enzyme activity is generated or increased by an interaction betweenthe first reporter (R1) and the second reporter (R2).
 9. The measurementmethod according to any of claims 1 to 4 wherein the material (A) andthe material (B) are antibodies or antibody-derived single chain Fv, Fv,a part of Fv, Fab′, Fab, or mutants thereof, each thereof recognizingdifferent sites in the subject material (X) to be measured.
 10. Themeasurement method according to any of claims 1 to 9 wherein thematerial (A), the material (B), the reporter (R1), the reporter (R2),the material (C), and the material (D) comprise peptidergic materials,and the first reagent and the second reagent comprise fusion proteins.11. The measurement method according to any of claims 1 to 10 whereinthe material (C) and the material (D) are both leucine zipper peptides.12. The measurement method according to any of claims 1 to 11 whereinthe measurement is carried out in a homogenous system in which the firstreagent and the second reagent are made to act simultaneously on thesubject material to be measured.
 13. The measurement method according toany of claims 1 to 11 wherein the measurement is carried out in aheterogeneous system in which, among the first reagent and the secondreagent, one of the reagents is immobilized in advance on a solid phase,and then the other reagent and the subject material to be measured aremade to act on the immobilized reagent.
 14. A measurement reagent foruse in the measurement method according to any of claims 1 to 13, themeasurement reagent including the material (A), the material (B), thereporter (R1), the reporter (R2), the material (C) and the material (D).15. A measurement kit that includes the measurement reagent according toclaim 14.